Dynamic phosphoproteomics reveals TORC1-dependent regulation of yeast nucleotide and amino acid biosynthesis

The effect of phosphorylation on metabolic enzyme activity could be inferred by correlating phosphoproteomics and metabolomics data. How phosphorylation regulates metabolism The phosphorylation events triggered directly by or downstream of the protein complex TORC1 enable yeast to adjust their metabolism to respond to changes in nutrient availability or nutritional quality. Oliveira et al. subjected yeast to changes in nutrient quality or to treatment with rapamycin, an inhibitor of TORC1. By temporally correlating changes in metabolite concentrations with phosphorylation events, they identified metabolic enzymes downstream of TORC1 and inferred the effect of phosphorylation on the activity of these enzymes, which included enzymes involved in nucleotide and amino acid metabolism and in carbohydrate storage. Phosphoproteomics studies have unraveled the extent of protein phosphorylation as a key cellular regulation mechanism, but assigning functionality to specific phosphorylation events remains a major challenge. TORC1 (target of rapamycin complex 1) is a kinase-containing protein complex that transduces changes in nutrient availability into phosphorylation signaling events that alter cell growth and proliferation. To resolve the temporal sequence of phosphorylation responses to nutritionally and chemically induced changes in TORC1 signaling and to identify previously unknown kinase-substrate relationships in Saccharomyces cerevisiae, we performed quantitative mass spectrometry–based phosphoproteomic analyses after shifts in nitrogen sources and rapamycin treatment. From early phosphorylation events that were consistent over at least two experimental perturbations, we identified 51 candidate and 10 known proximal targets of TORC1 that were direct substrates of TORC1 or of one of its kinase or phosphatase substrates. By correlating these phosphoproteomics data with dynamic metabolomics data, we inferred the functional role of phosphorylation on the metabolic activity of 12 enzymes, including three candidate TORC1-proximal targets: Amd1, which is involved in nucleotide metabolism; Hom3, which is involved in amino acid metabolism; and Tsl1, which mediates carbohydrate storage. Finally, we identified the TORC1 substrates Sch9 and Atg1 as candidate kinases that phosphorylate Amd1 and Hom3, respectively.